Epigenetic modifiers play vital roles in directing DNA structure, regulating gene expression, and determining disease-states. Scientists have recently taken interest in a epigenetic marker, 5-hydroxymethylcytosine (5hmC), which represents less than one percent of cytosines in the mammalian genome. Initially, 5hmC was thought primarily to be an intermediate in methylcytosine metabolism, but now the field is beginning to recognize 5hmC's involvement in a variety of cellular functions, even calling it the sixth base of the genome. One observation is that high local genomic concentrations of 5hmC have been linked to DNA recombination. Furthermore, the recombination protein Endonuclease G (Endo G) has been recently found to preferentially cleave 5hmC modified DNA over unmodified DNA. The proposed research aims to explore this link between 5hmC, Endo G, and recombination. The hypothesis is that 5hmC-modified DNA forms unique hydrogen bonds that promote pausing of a migrating DNA Holliday junction, and the paused junction provides a stable substrate for Endo G to bind. This model will be explored from a structural perspective with two specific aims. The first aim is to determine the structure and thermodynamics of 5hmC DNA using x-ray crystallography and differential scanning calorimetry. The second aim is to define the role of 5hmC on Endo G recognition by testing the binding of Endo G to a series of 5hmC, 5methyl-C, and unmodified-C duplex and junction DNA constructs. Overall, the mechanisms behind recombination aren't fully understood, and 5hmC is likely an important part the regulation process. Since recombination is an integral part of the DNA damage repair mechanism, 5hmC may help to target local DNA regions for repair. Particular cell types, including cancer cells, express lower levels of 5hmC. This suggests 5hmC levels are linked to the disease-state of cancer cells by de-regulating their DNA repair. This research will help build the fundamental base of knowledge for understanding recombination-related diseases. A complete understanding of 5hmC will furthermore help decode the mechanisms of genomic regulation, impacting more than just the recombination field.